Insulin is a key hormone required for metabolic homeostasis. Relative lack of insulin results in diabetes mellitus (DM), a condition marked by hyperglycemia and associated metabolic abnormalities. Long-term complications include retinopathy and nephropathy. Current insulin therapies are designed to achieve normoglycemia by mimicking pancreatic basal/bolus secretion of insulin. Classical clinical trials in Type 1 DM and new-onset Type 2 DM have shown that intensive treatment of blood glucose concentration leads to a significant reduction in microvascular complications at the cost of increased hypoglycemic events. We seek to design novel insulin analogs to enhance the safety and efficacy of insulin replacement therapy. Current insulin analog formulations (although representing a significant advance in the 1990s) are sub- optimal with respect to pharmacokinetics (PK) and physical stability. In brief, short-acting analogs are too slow relative to post-prandial pancreatic secretion whereas basal analogs do not offer true 24-hour PK. The goal of my thesis is to design and characterize a novel ultra-short-acting insulin analog for (i) prandial control in muli- injection regiments and (ii) use in a closed-loop system (smart pumps). The essential idea underlying my research plan is exploitation of an expanded genetic code to enable recombinant expression of non-standard insulin analogs. Preliminary studies of insulin analogs containing unnatural amino-acid substitutions (obtained by chemical synthesis) suggest the utility of a para-Cl-PheB24 (in place of PheB24) at the potential dimer interface of insulin lispro (the active component of HumalogR). We hypothesize that the chloro-aromatic substitution leads to accelerated disassembly of the zinc insulin analog hexamer due to the electronegativity or size of the halogen substitution (relative to Phe). Pilot studies in anesthetized pigs indicate that para-Cl-PheB24-insulin lispro exhibits accelerated pharmacodynamics relative to HumalogR. In my thesis I plan to express this non-standard analog in the yeast Pichia pastoris, determine its three-dimensional structure by X-ray crystallography, and characterize its physical stability under conditions of potential formulation. To my knowledge, this will be the first structure to be determined of a protein containing a chloro-aromatic substitution and the first example of how such a modification can enhance the therapeutic properties of a protein.